The main goal of this application is to test a potential host-directed therapeutic to enhance neutrophil microbial killing of extensively drug resistant (XDR) gram negative pathogens Klebsiella pneumoniae and Pseudomonas aeruginosa that are prevalent in ventilator-associated pneumonia (VAP). The emergence of XDR gram negative pathogens and the limited number of antimicrobial options present a rising challenge for the management of these patients. Enhancing the host immune response to fight infection may afford a novel, adjunct approach to conventional antimicrobials, particularly in critically ill hoss with waning immunity or immunosuppression that are often vulnerable to these pathogens. We have identified a novel mechanism by which thrombospondin-1 (TSP-1), a multifunctional extracellular matrix glycoprotein involved in cell-cell and cell-matrix interactions and released b a variety of cells during inflammation, restrains neutrophil microbial killing through inhibition o neutrophil serine protease (NSP) activity. Our preliminary data indicates that TSP-1 may provide a previously unrecognized, endogenous inhibitory mechanism to counter NSPs and curtail components of the microbial killing arsenal. Previous reports identified regions within the type II repeats domain of TSP-1 that show striking similarity to the consensus sequence found among the Kazal family of serine protease inhibitors. We have recently identified candidate small molecule inhibitors that may target a major cavity created within the type III repeats region of TSP-1. The major hypothesis of this application is that small molecule compounds that potentially disrupt TSP-1/neutrophil serine protease interaction can enhance microbial killing of XDR Klebsiella pneumoniae and Pseudomonas aeruginosa, which are two major VAP pathogens with few treatment options. The R21 is a proof-of-concept bench phase where we will examine whether small molecule compounds increase neutrophil microbial killing of XDR clinical isolates in vitro and in vivo using a mouse model. The R33 is the translational or bedside phase where we will examine whether TSP-1 based small molecule compounds will improve in vitro microbial killing of XDR pathogens utilizing neutrophils from suspected VAP patients. This approach may serve as the basis for a novel adjunct therapy to existing anti-microbial regimens that reduce bacterial burden in the lungs.